Jehan Waeytens

Research in Drug Development (RD3)

Microbiology, Bioorganic and Macromolecular Chemistry

Research activities

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Top ten recent articles

Articles dans des revues avec comité de lecture

2024

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as “functional amyloids”. In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

2023

Hfq C-terminal region forms a β-rich amyloid-like motif without perturbing the N-terminal Sm-like structure

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short β-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

New insights in the molecular regulation of the NADPH oxidase 2 activity: Negative modulation by Poldip2

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22phox, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393 cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2. Besides Nox4, the Nox1, Nox2, or Nox3 isoforms are also established partners of the p22phox protein raising the question of their regulation by Poldip2 and of the effect in cells expressing simultaneously different Nox isoforms. In this study, we have addressed this issue by investigating the potential regulatory role of Poldip2 on NADPH oxidase 2, present in phagocyte cells. Unexpectedly, the effect of Poldip2 on phagocyte NADPH oxidase 2 was opposite to that observed on NADPH oxidase 4. Using membranes from circulating resting neutrophils, the ROS production rate of NADPH oxidase 2 was down-regulated by Poldip2 (2.5-fold). The down-regulation effect could not be correlated to the interaction of Poldip2 with p22phox but rather, to the interaction of Poldip2 with the p47phox protein, one of the regulatory proteins of the phagocyte NADPH oxidase. Our results show that the interaction of Poldip2 with p47phox constitutes a novel regulatory mechanism that can negatively modulate the activity of NADPH oxidase 2 by trapping the so-called “adaptor” subunit of the complex. Poldip2 could act as a tunable switch capable of specifically regulating the activities of NADPH oxidases. This selective regulatory role of Poldip2, positive for Nox4 or negative for Nox2 could orchestrate the level and the type of ROS generated by Nox enzymes in the cells.

Determination of Secondary Structure of Proteins by Nanoinfrared Spectroscopy

Nanoscale infrared spectroscopy (AFMIR) is becoming an important tool for the analysis of biological sample, in particular protein assemblies, at the nanoscale level. While the amide I band is usually used to determine the secondary structure of proteins in Fourier transform infrared spectroscopy, no tool has been developed so far for AFMIR. The paper introduces a method for the study of secondary structure of protein based on a protein library of 38 well-characterized proteins. Ascending stepwise linear regression (ASLR) and partial least square (PLS) regression were used to correlate spectrum characteristic bands with the major secondary structures (α-helixes and β-sheets). ASLR appears to provide better results than PLS. The secondary structure predictions are characterized by a root mean square standard error in a cross validation of 6.39% for α-helixes and 6.23% for β-sheets.

2022

Thermochemical Liquefaction of Pomace Using Sub/Supercritical Ethanol: an Integrated Experimental and Preliminary Economic Feasibility Study

Fossil sourced chemicals such as aromatics, are widely employed in the chemical industry for the production of commodity items. Recognizing the un-sustainability of existing approaches in the production of these chemicals, the current study investigated the valorization of apple pomace (AP) for their production. The present study assessed AP valorization by imposing variations in processing conditions of temperature (100-260 °C), time (0.5-12 h), alcohol/water ratio v/v (0:1-1:0), and Fe3+/H2O2 molar ratio (10:1-100-1), in accordance to the Box-Behnken experimental design. The optimal yield of the oil was 24.6 wt.%, at the temperature, time, alcohol/water ratio v/v, and Fe3+/H2O2 molar ratio of 260 °C, 4.7 h, 1, and 100, respectively. Notably, the application of gas chromatography-mass spectroscopy showed that the oil product contained mainly aromatics and interestingly also alkanes, indicating that the experimental conditions imposed promoted secondary hydrogenation reactions of oxygen-containing species during AP valorization. A consideration of the comparative economics of the proposed AP valorization and the existing AP management approach, using approximate estimation techniques, highlighted the potential of a ~ 59% reduction in the unit cost of AP management. The study therefore presents a compelling basis for future investigations into AP waste management using the thermochemical liquefaction technology. Graphical abstract: [Figure not available: see fulltext.].

Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq

Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.

Plastic biodegradation: Do Galleria mellonella Larvae Bioassimilate Polyethylene? A Spectral Histology Approach Using Isotopic Labeling and Infrared Microspectroscopy

Environmental pollution by the nearly nonbiodegradable polyethylene (PE) plastics is of major concern; thus, organisms capable of biodegrading PE are required. The larvae of the Greater Wax Moth, Galleria mellonella (Gm), were identified as a potential candidate to digest PE. In this study, we tested whether PE was metabolized by Gm larvae and could be found in their tissues. We examined the implication of the larval gut microbiota by using conventional and axenic reared insects. First, our study showed that neither beeswax nor LDPE alone favor the growth of young larvae. We then used Fourier transform infrared microspectroscopy (μFTIR) to detect deuterium in larvae fed with isotopically labeled food. Deuterated molecules were found in tissues of larvae fed with deuterium labeled oil for 24 and 72 h, proving that μFTIR can detect metabolization of 1 to 2 mg of deuterated food. Then, Gm larvae were fed with deuterated PE (821 kDa). No bioassimilation was detected in the tissues of larvae that had ingested 1 to 5 mg of deuterated PE in 72 h or in 19 days, but micrometer sized PE particles were found in the larval digestive tract cavities. We evidenced weak biodegradation of 641 kDa PE films in contact for 24 h with the dissected gut of conventional larvae and in the PED4 particles from excreted larval frass. Our study confirms that Gm larvae can biodegrade HDPE but cannot necessarily metabolize it.

2021

Molecular mechanism of thiamine pyrophosphate import into mitochondria: a molecular simulation study

The import of thiamine pyrophosphate (TPP) through both mitochondrial membranes was studied using a total of 3-µs molecular dynamics simulations. Regarding the translocation through the mitochondrial outer membrane, our simulations support the conjecture that TPP uses the voltage-dependent anion channel, the major pore of this membrane, for its passage to the intermembrane space, as its transport presents significant analogies with that used by other metabolites previously studied, in particular with ATP. As far as passing through the mitochondrial inner membrane is concerned, our simulations show that the specific carrier of TPP has a single binding site that becomes accessible, through an alternating access mechanism. The preference of this transporter for TPP can be rationalized mainly by three residues located in the binding site that differ from those identified in the ATP/ADP carrier, the most studied member of the mitochondrial carrier family. The simulated transport mechanism of TPP highlights the essential role, at the energetic level, of the contributions coming from the formation and breakage of two networks of salt bridges, one on the side of the matrix and the other on the side of the intermembrane space, as well as the interactions, mainly of an ionic nature, formed by TPP upon its binding. The energy contribution provided by the cytosolic network establishes a lower barrier than that of the matrix network, which can be explained by the lower interaction energy of TPP on the matrix side or possibly a uniport activity.

Use and misuse of FTIR spectroscopy for studying the bio-oxidation of plastics

Experimental Characterization of Commercial and Synthesized Aromatic Polyamide Films for Reverse Osmosis Membranes

The complex structure of the active aromatic polyamide (APA) layer of reverse osmosis membranes needs to be precisely described for understanding and predicting solute rejection. A commercial reverse osmosis membrane (CPA2, Hydranautics) was chosen as a case study, and home-made APA films were prepared by free-standing interfacial polymerization of trimesoyl chloride (TMC) and m-phenylenediamine (MPD) at different temperatures to obtain different film structures. Their morphology, thickness, void fraction, density, and chemical composition were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), profilometry, ellipsometry, dynamic vapor sorption (DVS), and X-ray photoelectron spectroscopy (XPS). All samples have a multilevel topological structure as a dense base upon which generate a valley-ridge structure for CPA2 and a chimneylike structure for synthesized samples, whose thicknesses increase with temperature. An average void fraction of 35% was deduced for the APA from CPA2, allowing the calculation of a dry density of its dense phase of 1.24 g·cm-3, which is rarely investigated for commercial membranes. New chemical descriptors considering chain heads, tails, and branches were introduced to improve the interpretation of chemical composition results. Being more realistic than the cross-linking degree, they suggested for the CPA2 active layer a back surface chemically less cross-linked than the top surface.

Probing amyloid fibril secondary structures by infrared nanospectroscopy: Experimental and theoretical considerations

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillary structure with a higher β-sheet content than their native structure. Attenuated total reflection Fourier transform infrared spectroscopy only provides bulk analysis of a sample therefore it is impossible to discriminate between different aggregated structures. To overcome this limitation, near-field techniques like AFM-IR have emerged in the last twenty years to allow infrared nanospectroscopy. This technique obtains IR spectra with a spatial resolution of ten nanometres, the size of isolated fibrils. Here, we present essential practical considerations to avoid misinterpretations and artefacts during these analyses. Effects of polarization of the incident IR laser, illumination configuration and coating of the AFM probes are discussed, including the advantages and drawbacks of their use. This approach will improve interpretation of AFM-IR spectra especially for the determination of secondary structures of species not accessible using classical ATR-FTIR.

2020

Characterization by Nano-Infrared Spectroscopy of Individual Aggregated Species of Amyloid Proteins.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillar structure with a higher β-sheet content than in their native structure. To characterize them, we used an innovative tool that coupled infrared spectroscopy with atomic force microscopy (AFM-IR). With this method, we show that we can detect different individual aggregated species from oligomers to fibrils and study their morphologies by AFM and their secondary structures based on their IR spectra. AFM-IR overcomes the weak spatial resolution of usual infrared spectroscopy and achieves a resolution of ten nanometers, the size of isolated fibrils. We characterized oligomers, amyloid fibrils of Aβ42 and fibrils of α-synuclein. To our surprise, we figured out that the nature of some surfaces (ZnSe) used to study the samples induces destructuring of amyloid samples, leading to amorphous aggregates. We strongly suggest taking this into consideration in future experiments with amyloid fibrils. More importantly, we demonstrate the advantages of AFM-IR, with a high spatial resolution (≤ 10 nm) allowing spectrum recording on individual aggregated supramolecular entities selected thanks to the AFM images or on thin layers of proteins.

Nanometric chemical speciation of abnormal deposits in kidney biopsy: Infrared-nanospectroscopy reveals heterogeneities within vancomycin casts.

Dipeptide repeat derived from C9orf72 hexanucleotide expansions forms amyloids or natively unfolded structures in vitro

2019

The bacterial amyloid-like Hfq promotes in vitro DNA alignment

The Hfq protein is reported to be involved in environmental adaptation and virulence of several bacteria. In Gram-negative bacteria, Hfq mediates the interaction between regulatory noncoding RNAs and their target mRNAs. Besides these RNA-related functions, Hfq is also associated with DNA and is a part of the bacterial chromatin. Its precise role in DNA structuration is, however, unclear and whether Hfq plays a direct role in DNA-related processes such as replication or recombination is controversial. In previous works, we showed that Escherichia coli Hfq, or more precisely its amyloid-like C-terminal region (CTR), induces DNA compaction into a condensed form. In this paper, we evidence a new property for Hfq; precisely we show that its CTR influences double helix structure and base tilting, resulting in a strong local alignment of nucleoprotein Hfq:DNA fibers. The significance of this alignment is discussed in terms of chromatin structuration and possible functional consequences on evolutionary processes and adaptation to environment.

Evaluating Mechanical Properties of Polymers at the Nanoscale Level via Atomic Force Microscopy-Infrared Spectroscopy

Probing the Metal-Oxide/Polymer Molecular Hybrid Interfaces With Nanoscale Resolution Using AFM-IR

2016

Caractérisation de polymères par analyse infrarouge à l'échelle nanométrique

AFM-IR is a recent technique based on the detection of IR absorption by the tip of an AFM. It provides local spectroscopic information at subdiffraction resolution. In this work, model samples made of polystyrene thin films, as well as commercial multilayer packaging films have been characterised by AFM-IR. Infrared spectra with sufficient signal to-noise ratio (S/N) were recorded in a single scan for thicknesses down to 30 nm. Increasing the number of scans, and hence the S/N, thinner samples (16 nm) could be analysed. AFM-IR is particularly powerful for multilayer samples, and it was possible to identify the chemical nature of each layer in the commercial sample, as well as the typical interfacial thickness between each layer. A value of 600 nm was determined, which would have been inaccessible by conventional IR microscopy. In addition to topographic and spectroscopic information, it is proposed on an experimental basis that the deflection profile of the AFM tip upon relaxation from the photothermally induced resonance could be used to extract some viscoelastic properties of the sample, by analogy with the widespread contact resonance AFM.